快速鉴定猪链球菌和马链球菌兽疫亚种双重PCR方法的建立

Establishment of Double PCR for Rapid Identification of Streptococcus suis and Streptococcus equi Subspecies

本试验旨在建立一种快速、特异、敏感的双重PCR鉴定猪链球菌和马链球菌兽疫亚种病原检测方法。根据猪链球菌GDH蛋白和马链球菌类M蛋白的基因保守区分别设计引物,优化了该双重PCR检测方法的引物浓度及比例,并筛选了其最佳退火温度;用该双重PCR反应体系以其他几株阴性菌株为对照,检测了该反应体系的特异性。以新鲜培养的猪链球菌倍比稀释后进行菌落计数,对该检测方法的敏感性进行了鉴定。M-like和GDH引物的加入量均为1μL(20pmol/L),最佳退火温度为52.3℃;该双重PCR反应体系有较高敏感性,检测马链球菌兽疫亚种和猪链球菌的敏感度分别达100和10CFU;特异性试验结果显示,常见的5种病原菌在该双重PCR体系中无特异性条带出现;临床应用该方法分离鉴定了1株猪链球菌和2株马链球菌兽疫亚种。本试验建立了一种能同时检测猪链球菌和马链球菌兽疫亚种的双重PCR方法,且该方法应用快速、特异且敏感。

To establish a rapid, specific and sensitive double PCR identification method of Streptococcus suis and Strepto- coccus equi subspecies pathogen,primers were designed based on gene conserved region of the M-like protein of Streptococcus equi subspecies and GDH protein of Streptococcus suis. The amount of M-like and GDH primers were both 1 μL （20 pmol/L）. The optimal annealing temperature was 52. 3 ℃. The sensitivity of double PCR were 10 CFU for Streptococcus suis and 100 CFU for Streptococcus equi subspecies. The specific trials had shown that five kinds of common pathogens had non-specific bands appeared in the double PCR system. 1 Streptococcus suis and 2 Streptococcus equi subspecies were detected by this method.